Protein ID (NP or XP #) or Wolbachia#: XP # 844733.1 Organism (including strain): Trypanosoma brucei brucei Etiologic Risk Group: Risk Group 2 Background/Disease Information: Trypanosoma brucei is a protozoan with flagella that causes African Sleeping Sickness (African trypanosomiasis) in humans. The disease is endemic in regions of sub-Saharn Africa. Symptoms occur in two stages. The first stage is characterized by fever, headaches, joint pains, and itching. Invasion of the circulatory and lymphatic systems by the parasites can result in swelling of the lymph nodes. The second stage, or neurological phase, is caused by the invasion of the central nervous system by the parasite. This results in disruption of the sleeping cycle. Infected individuals experience a disorganized and fragmented sleep-wake cycle. The tsetse fly serves as both host and vector for trypanosoma parasites. Essentiality of this protein: Essential, significant loss of fitness in blood stream forms has been determined. Used for cytokinesis Complex of proteins?: Forms complex with regulatory sub-units GM and GL. Druggable Target: 0.6
- Now 0.3 Druggability (082413 - Dr .B)
Structure Available (PDB or Homology model) -- Closest PDB entry using homology model: 2O8A -- Homology Model option: ----
Fig. 1. Pairwise alignment of BLASTP search in NCBI of T. brucei protein phosphatase 1 against the PDB
---- Query Coverage: 303 ---- Max % Identities: 76% ---- % Positives: 89% ---- Chain used for homology: Chain A
Current Inhibitors: Calyculinamide A , Calyculin A, C1/C34-Calyculin A, Des-N-methylcalyculin A, Calyculin A 21-acetate, Hemicalyculin A, Calyculin C, Calyculin F, Calyculin J, Calyculin B, Calyculin E, Dephosphonocalyculin A, Calyculin A 11,13,21-triacetate, 11, 13-O-isopropylidene-calyculin A, C9/C35 calyculin
Image of protein (PyMol with features delineated and shown separately):
Fig. 2. Crystal structure of PP1
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSVDVDAIIDKLLEVRLSKPGKQVSLSENDVKNLVMRSREILLSQPALLELEAPIKICGD
IHGQYYDLIRLFDNGGFPPSANYLFLGDYVDRGKQGLETICLVLAFKVKFPENFFILRGN
HECASINRIYGFFDECKRRYNIRLWKVFTDTFNCLPVACIIDDKIFCCHGGLSPDLQSME
QIKKIERPCDVADTGLICDLLWSDPEEGLSGWGENDRGVSYTFGQDIVAKFLSRHDFDLI
VRAHQVVEDGYQFFATRQLITIFSAPNYCNEFDNSGAVMSVDADLLCSFQILKPSVKKPK
YFQ length of your protein in Amino Acids: 303 amino acids Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 34443.5 kDa Molar Extinction coefficient of your protein at 280 nm wavelength: 32150 TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only): *GC% Content for gene (codon optimized): Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers): (link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol) -- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):
NCBI Gene # or RefSeq#: 3657166
Tb927.4.5030
Protein ID (NP or XP #) or Wolbachia#: XP # 844733.1
Organism (including strain): Trypanosoma brucei brucei
Etiologic Risk Group: Risk Group 2
Background/Disease Information:
Trypanosoma brucei is a protozoan with flagella that causes African Sleeping Sickness (African trypanosomiasis) in humans. The disease is endemic in regions of sub-Saharn Africa. Symptoms occur in two stages. The first stage is characterized by fever, headaches, joint pains, and itching. Invasion of the circulatory and lymphatic systems by the parasites can result in swelling of the lymph nodes. The second stage, or neurological phase, is caused by the invasion of the central nervous system by the parasite. This results in disruption of the sleeping cycle. Infected individuals experience a disorganized and fragmented sleep-wake cycle. The tsetse fly serves as both host and vector for trypanosoma parasites.
Essentiality of this protein: Essential, significant loss of fitness in blood stream forms has been determined. Used for cytokinesis
Complex of proteins?: Forms complex with regulatory sub-units GM and GL.
Druggable Target: 0.6
- Now 0.3 Druggability (082413 - Dr .B)
EC#: 3.1.3.16
Link to BRENDA EC# page:
-- http://www.brenda-enzymes.info/literature/lit.php4?e=3.1.3.16&r=655670
Enzyme Assay information:
-- http://www.sigmaaldrich.com/etc/medialib/docs/Sigma/Enzyme_Assay/calcineurin.Par.0001.File.tmp/calcineurin.pdf
-- http://www.brenda-enzymes.org/php/result_flat.php4?ecno=3.1.3.16
-- List cost and quantity of substrate reagents and supplier: pNPP, $79.99 for 100 mL
Structure Available (PDB or Homology model)
-- Closest PDB entry using homology model: 2O8A
-- Homology Model option:
----
---- Query Coverage: 303
---- Max % Identities: 76%
---- % Positives: 89%
---- Chain used for homology: Chain A
Current Inhibitors: Calyculinamide A , Calyculin A, C1/C34-Calyculin A, Des-N-methylcalyculin A, Calyculin A 21-acetate, Hemicalyculin A, Calyculin C, Calyculin F, Calyculin J, Calyculin B, Calyculin E, Dephosphonocalyculin A, Calyculin A 11,13,21-triacetate, 11, 13-O-isopropylidene-calyculin A, C9/C35 calyculin
Expression Information: Has been expressed in E. coli
Purification Method: Western and Northern Blot analysis
http://www.ncbi.nlm.nih.gov/pubmed/?term=T.+brucei+protein+phosphatase+1+expression
Image of protein (PyMol with features delineated and shown separately):
Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MSVDVDAIIDKLLEVRLSKPGKQVSLSENDVKNLVMRSREILLSQPALLELEAPIKICGD
IHGQYYDLIRLFDNGGFPPSANYLFLGDYVDRGKQGLETICLVLAFKVKFPENFFILRGN
HECASINRIYGFFDECKRRYNIRLWKVFTDTFNCLPVACIIDDKIFCCHGGLSPDLQSME
QIKKIERPCDVADTGLICDLLWSDPEEGLSGWGENDRGVSYTFGQDIVAKFLSRHDFDLI
VRAHQVVEDGYQFFATRQLITIFSAPNYCNEFDNSGAVMSVDADLLCSFQILKPSVKKPK
YFQ
length of your protein in Amino Acids: 303 amino acids
Molecular Weight of your protein in kiloDaltons using the Expasy ProtParam website: 34443.5 kDa
Molar Extinction coefficient of your protein at 280 nm wavelength: 32150
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
CDS Gene Sequence (paste as text only):
ATGTCACAGGAGGGTGGGAAATCAGAACCGGACGCCAACGAAAAAGAGCAACGGAACCAAAAATTAAGGC
TAGGCCTTGAGGCCATGTTCTGTGCTCAAGAGATACCTTCTGAGAAACGCACGGACATTGAAAGTGACGG
CGAGGAAGTGCCTCCAAGCATTACTCGAGATGAAGATAGATACCGTCAGGTGGCGGAGGCTATTCTTAGG
ATTGATACGAAGAGCAAAATCATAGAGATAAATAATATTCGCCTGTTTTCAGTGGCTGAAATTGAGCTTG
ACAAGTTGACTGAATGCACATCCCTTTCCTTGCGCAAGAATCTCTTGCATGATCTTATTCCGTTTCCTGA
GGACCTCGCCGATCGCCTTGATGAGTTGGATCTTTTCGACAACAAAATTCGTAAGCTGAACGATTTTTTC
GAGACAGTAACAGTCCCTGGTGACCCGCCCACGACGAAAACACTCCCCAACGCATACAAATGTCTCACGA
AATTGGACCTAAGTTACAATCAGATTCGTGAAATCGGTGGACTCGATTCCATCGGGGGTACGCTGAGAGA
GCTTTATCTGGTCGAAAATAAAATTAAAGAGGTTAAAAATCTTGATTCTCTGGTGAACCTTGAGTTACTT
GAATTAGGGGGCAACCGTTTACGTGCCATCGGTTCCGGGTTGGAGAAGTTGACGAAGTTGAAACAGTTGT
GGCTCGGCAAGAATAAGATAAGTTCCATTGGTACCGCGCTTCACAAATTGGTTTCGCTAGAGATCCTTAG
TTTGCAGGCAAACCGAATTACCTCTGTTGATGCGGAAAACTTCTTGGGAGCAAAGGCCAATCCTAATTTG
AGGGAAGTATACTTGTCCGAGAACGGCCTTACTTCTGTTGGGAACGTGCGACACCTATCAACAATCAAAA
TTATCGATTTTAGTTTCAACTCGATATGTTCAATCGACGCTGAAGAGATCAATCCGCAAACCATGCCCAA
ACTTGAGGAGTTTTGGCTCACTGATGGAAATATAGCAGATTGGGAAGAGGTAGGAAAGTTAAGTGGCTTC
ACAAGCACTTTGAAGACCGTGTATCTTGAGCGTAACCCAATTGAAGAAGATAAGAGGTACCGCGACAAGG
TATACATGTACCTGCCCTTCTTGGTACAGATTGATTCATGGCCAATTGTGAATAAGGGAAATCTTGAAGC
TGATCGTAAGCGAAAAGCTTGA
GC% Content for gene: 44.6%
*CDS Gene Sequence (codon optimized) - copy from output of Primer Design Protocol (paste as text only):
*GC% Content for gene (codon optimized):
Primer design results for pNIC-Bsa4 cloning (list seqeunces of all of your ~40 nt long primers):
(link to DNA Works output text file - that should be saved in your Google Docs folder after you did the primer design protocol)
-- Ask a mentor, Dr. B, or a fellow researcher -how to link a GDocs file if you are not sure how to.
Primer design results for 'tail' primers (this is just 2 sequences):